1. Field of the Invention
This invention relates to fluorescent methods of detecting blood, and specifically relates to the use of fluorescein for detection of latent bloodstains on solid surfaces.
2. Relevant Prior Art
In forensic investigations, detection of blood often provides critical information about commission of a crime. Because criminals often attempt to hide evidence of criminal activity, blood is often wiped or washed away from a crime scene. Therefore, detection of latent bloodstains may be the only means of discovering this type of information related to the crime committed.
One method of detecting faint or dilute bloodstains includes spraying the area suspected of containing bloodstains with a chemiluminescent agent (5-amino-2,3-dihydro-1,4-phthalazine, also known as luminol) and then detecting emitted light where the blood is located. Oxidation of luminol is accompanied by a striking emission of light that can be visually or photographically detected. Oxidation of luminol occurs in the presence of iron and peroxides, both of which are generally present in bloodstains. Because of this property, luminol has been one of the most commonly used bloodstain detection reagents. Luminol can also be oxidized by environmentally-present iron, copper, cyanides and peroxides, which can result in high background signals when attempting to locate latent bloodstains in some environments.
In addition, luminol has been characterized as a "possible carcinogenic" on material safety data sheets (MSDS) provided by commercial suppliers to provide consumers with safety information. Because of increased concern by users and regulatory agencies, such as the Environmental Protectional Agency, with respect to toxic and/or mutagenic reagents used in the work place, there is a need for an alternative to luminol for detection of latent bloodstains. Enforcement of more stringent safety guidelines may also have limited the use of luminol. Thus, there is a need for a safe, reliable alternative bloodstain enhancement technique.
Fluorescein (3',6'-dihydroxyspiro[isobenzofuran-1 (3H), 9'-[9H]xanthen]-3-one) and related compounds have been used as fluorescent agents in clinical diagnostics for many years. Fluorescein may be used to detect and locate the position of gastrointestinal bleeding (e.g., see U.S. Pat. No. 3,483,859). U.S. Pat. No. 4,341,223 discloses a fluoresceable composition that includes fluorescein as a tracer for use in the vascular system, particularly for detecting ophthalmic conditions. Fluorescein has been used in angiography diagnostics for vascular ophthalmic disorders (Reichel, E., & Puliafito, C., "Indocyanine Green (ICG) angiography in the diagnosis and treatment of choroidal neovascularization," Clinical Modules, New England Eye Center, January 1994). Fluorescein has been combined with other reagents to measure the circulating red cell volume, the total blood volume and red cell survival as disclosed in U.S. Pat. No. 5,536,643. A fluorescein fatty acid monoester has been used diagnostically, where fluorescein released in urine or serum provides a measurement of exocrine pancreatic function as disclosed in U.S. Pat. No. 3,786,140. Fluorescent cell stains have also been used in in vitro diagnostics, such as disclosed in U.S. Pat. No. 4,345,027.
Fluorescein has been widely used in detecting activities associated with biological samples, including blood, and has been suggested as an alternative reagent to replace luminol for detecting bloodstains (Maucieri, L. & Monk, J., "Enhancement of Faint and Dilute Bloodstains With Fluorescence Reagents," Cal. Assoc. Criminalists, Summer 1992). In this process, a fluorescin (2-(3,6-dihydroxy-9H-xanthen-9-yl)-benzoic acid) solution is applied to the bloodstain, and is thereby converted to fluorescein by the peroxide and heme of the blood, to produce a fluorescence. The method may include hydrogen peroxide treatment of the fluorescintreated area to enhance the fluorescence signal. The fluorescein method for detecting latent bloodstains at suspected crime scenes, however, generally has not provided the resolution desired for documentation of evidence when blood has been detected. That is, although the presence of blood may be detected, detail in the pattern of the stain is generally lost due to running and smearing of the stain during processing, particularly if the stain is on a relatively non-porous and vertical surface. Hence, there is a need for a method of detecting latent bloodstains that does not require the use of luminol, that provides sensitivity for detecting small amounts of blood on a variety of surfaces, and that provides sufficient resolution to detect pattern characteristics and details of latent bloodstains.
In addition, production of the fluorescin solution used in the fluorescein detection method involves relatively complex chemical procedures (i.e., a refluxing reaction) that can only be performed by those with sufficient training and equipment. Because a relatively fresh fluorescin solution is required to practice a fluorescein detection method, this limits use of the method to those facilities that have the capacity to perform the chemical steps needed to produce the reagent. Thus, there is also a need for a relatively simple method of producing fluorescin reagent.